Lyme borreliosis (Lyme disease), a systemic illness with a wide spectrum of clinical symptoms, was named for Lyme, Conn., where the disease was identified. Although subsets of the diverse clinical manifestations of Lyme disease were recorded in Europe early in this century, recognition of the disease as a distinct clinical entity did not occur until the mid-1970s. Today, Lyme disease is the most common tickborne zoonosis in the United States, with more than 6000 human infections reported each year.
Lyme disease is a multisystem disorder with dermatologic, neurologic and musculoskeletal components that is caused by the spirochete Borrelia burgdorferi. The risk of a human acquiring Lyme disease is dependent on an interplay of microbial, environmental, and demographic factors. Ultimately, transmission is effected by nymphal ticks of the Ixodes ricinus complex. Illness usually develops three to thirty days following the tick bite, and often begins with a primary skin lesion called erythema migrans, followed by cardiac, neurologic, or arthritic symptoms. These resulting symptoms vary in severity, are disease stage dependent, and often mimic other conditions. This multifaceted presentation often delays and confuses clinical diagnosis.
Currently bacterial culture and serologic methods are used in diagnosis. See, A. C. Steere, N. Engl. J. Med., 321, 568-596 (1989). In the early stages of Lyme disease, B. burgdorferi can be readily recovered by culture from biopsy specimens of the erythema migrans skin lesions. P. D. Mitchell et al., Am. J. Clin. Pathol., 99, 104 (1993). However, as the disease progresses, the organism becomes increasingly difficult to detect by culture. In addition, limited sensitivity and specificity and lack of test standardization between laboratories have hindered the interpretation of results.
The inadequacy of current diagnostic techniques is well-illustrated by the difficulties encountered in confirming diagnosis in patients with suspected Lyme arthritis, a late manifestation of Lyme disease that results in episodic synovial inflammation and swelling. In these patients, successful cultivation of spirochetes from synovial (joint) fluid specimens has been reported only twice. Lyme arthritis can usually be treated successfully with either a one-month course of doxycycline or amoxicillin or a two-week course of intravenous certriaxone or penicillin. In some patients, however, arthritis persists despite multiple courses of oral and intravenous antibiotic therapy. It has been unclear whether this treatment-resistant course results from persistent infection or from postinfective immune-mediated phenomena. The ability to demonstrate unequivocally the presence or absence of B. burgdorferi in the joint would improve the understanding of the pathogenesis of Lyme arthritis, and assist in identifying appropriate treatment protocols.
Detection of deoxyribonucleic acid (DNA) from B. burgdorferi in tissue and fluid specimens using the polymerase chain reaction (PCR) provides direct proof of continuing infection and is an important diagnostic tool. See, D. H. Persing, "Molecular detection of Borrelia burgdorferi," in S. Schutzer, ed. Lyme Disease: Molecular and Immunologic Approaches, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press, 299-315 (1993). To date, genomic and plasmid DNA sequences from B. burgdorferi have been used as targets with limited success. For example, specific DNA sequences encoding the outer surface protein A (OSPA) present on the 54-kb plasmid in B. burgdorferi B31, along with the genomic sequences encoding the flagellin and 16S rDNA genes have been used as targets. See, D. H. Persing et al., Science, 249, 1420-1423 (1990); D. H. Persing et al., J. Infect. Dis., 169, 668-672 (1994); S. L. Goodman et al., Infect. Immun., 59, 269-278 (1991); and P. A. Rosa et al., J. Infect. Dis., 160, 1018-1029 (1989). The range and quality of specimen types and collection and transport conditions have confounded attempts to design a single efficient standard processing technique; the varied physical characteristics and DNA content of the specimens, ranging from bacterial cultures, tick extracts, whole blood, serum, joint fluid, urine, and cerebrospinal fluid, require that protocols be developed for each specimen type to obtain a satisfactory yield of target DNA free from inhibitors that are often present. Moreover, genomic B. bergdorferi DNA is considerably more difficult than extrachromosomal DNA to detect in advanced cases of Lyme disease. D. H. Persing et al., J. Infect. Dis., 169, 668-672 (1994). Furthermore, existing methods for detecting B. burgdorferi DNA using PCR are strictly experimental and have no proven clinical value. Thus, what is needed is a highly selective, specific, sensitive, and practical method for detecting genetic evidence of the Lyme-disease causing spirochete in a variety of clinical specimens at varying stages of the disease.